The results of nitric oxide (NO) infusions in normal volunteers and NO infusions and inhalation in experimental animals confirms that NO can be transported as a hormone and thus has the potential to be a pharmacological agent (i.e., a drug). We believe that the lack of vascular effects in our sickle cell patients is due to the presence of circulating hemoglobin and that this contributes to the pathophysiology of this and other chronic and acute hemolytic syndromes, especially the pulmonary hypertension complications which we have found to be severe and of high frequency in older patients. We also find that NO-bioactivity destruction appears to occur in tissues to a much greater extent than in the vascular bed. We have also been fortunate in being able to use the Critical Care Medicine Department canine facility and test inhaled nitrite and nitrate ions in hypoxic dogs to see if administering NO bioactivity in this way would be therapeutically possible. We tested nitrate as here is evidence that pulmonary tissue has some levels of enzymes that can reduce nitrate ions. We have found strong effects of nitrite inhalation on both pulmonary and systemic parameters, as others have reported for other animal models, but the effects of nitrate are only seen intermittently, suggesting that nitrate in the salivary fluid is reduced to nitrite and then systemic and pulmonary exposure give NO effects due to nitrite reduction processes. These results are now being prepared for publication. Detailed metabolic studies of nitrate-nitrite-NO metabolism in animals and people require tracer studies which may be approached using non-toxic heavy isotopes. Our collaborators in Newcastle are developing protocols to do this in animals and then perhaps in human subjects. The have sent us samples from various animal ingestion studies and we are using our highly sensitive and accurate chemi-luminescence methods to quantity nitrate and nitrite levels in these blood samples. (One of their staff visited our laboratory for several weeks to learn how to do these assays.) Our collaborators in Sao Paulo, Brazil have much experience in the pharmacology of NO and we are working with them on various animal models of hypertension with the hope of eventual clinical studies. Our collaborators in Bangkok have administered low levels of nitrite to thalassemia patients and we have worked with them to show that VASP phosphorylation is affected, as a parameter for measuring platelet inhibition (as described in DK 025093-200. Lastly we have established a collaboration with a muscle physiology group in Exeter, UK and we have been measuring the levels of nitrate at rest and with exercise - in the human tissues to see if our animal results obtain for human subjects, as other groups have now reported. We believe that muscle levels of nitrate must be considered in dietary interventions, for pharmacological effects or for affecting performance. Two new projects have very recently been initiated. First, we are studying the mechanism of nitroglycerine reduction as our recent work has suggested the possibility of new pathways that might enhance clinical utility. Second, we have been measuring nitrate and nitrite in whole rodent eyes as a beginning of measuring these levels in various compartments of the eye in larger animal models and perhaps in human samples.